Production of virus and purification of viral envelope proteins for vaccine use

ABSTRACT

Immunogenic envelope glycoproteins are produced from enveloped virus, such as of the paramyxoviridae family, particularly PIV-3 and RSV, by culturing the virus in the substantial absence of exogenous serum proteins, isolating the virus from the tissue culture, solubilizing the envelope glycoproteins and isolating the solubilized envelope glycoproteins by chromatography.

This is a continuation of application Ser. No. 07/773,949 filed Jun. 28,1990.

FIELD OF INVENTION

The present invention relates to the preparation and purification ofenvelope glycoproteins, particularly from the Paramyxoviridae family ofhuman pathogenic viruses. The present invention also relates to theformulation of a mixture of the purified glycoproteins to give anefficacious and safe vaccine for use in infants and young children toprotect against the diseases caused by the viruses.

BACKGROUND OF THE INVENTION

Human Parainfluenza type 3 (PIV-3) and Respiratory Syncytial subgroups Aand B (RSV-A,B) viruses, which are members of the Paramyxoviridae, havebeen identified as major pathogens responsible for severe respiratorydisease in infants and young children. It has been shown that bothformaldehyde-inactivated and live-attenuated vaccines failed to provideadequate protection against these diseases in clinical trials.Currently, safe and effective vaccines for prevention against theseviral infections are not available. Thus, the development of efficaciousPIV-3 and RSV vaccines is urgently required.

The major immunogenic proteins of RSV and PIV-3 have been identified,thereby providing the scientific rationale for a sub-unit approach tovaccine development. It has been shown that an in vivo protectiveresponse is contingent on the induction of neutralizing antibodiesagainst the major viral surface glycoproteins. For PIV-3, theseprotective immunogens are the HN protein, which has a M.W. of 72 kDa,and possesses both hemagglutinin and neuraminidase activities and the F(fusion) protein, which has a M.W. of 65 kDa, and is responsible forboth fusion of the virus to the host cell membrane and cell-to-cellspread of the virus. Immunogenicity studies in hamsters have shown thatantibodies to both HN and F proteins were essential for protectionagainst challenge with PIV-3. In addition, the presence of antibodies toboth envelope glycoproteins was reported to correlate with protection inchildren naturally infected with PIV-3. For RSV, the two immunogenicsurface glycoproteins are the 80-90 kDa glycoprotein (G) and the 70 kDafusion (F) protein. The G and F proteins are thought to be functionallyanalogous to the PIV-3 HN and F proteins, respectively. In humans,antibodies to both PIV-3 viral surface glycoproteins are necessary forprotection against PIV-3 infection, whereas anti-fusion proteinantibodies are sufficient to elicit a cross-protective response againstRSV infection.

SUMMARY OF INVENTION

In accordance with the present invention, the inventors have found aprocess for the production and purification of both PIV-3 and RSVviruses as well as a procedure for the purification of viral envelopesurface glycoproteins generally. This process results in preparationsthat are potent PIV-3 immunogens in experimental animals and may beacceptable for use as vaccines in children. This procedure also isdirectly applicable for the production of virus and the purification ofsurface glycoproteins from any of the enveloped viruses, such asinfluenza, in which the major envelope proteins are important ineliciting an immunogenic response. The invention also includes thehighly-purified immunogenic glycoproteins.

DESCRIPTION OF INVENTION

In the present invention, enveloped viruses, such as PIV-3 and RSVviruses, are grown in tissue culture on cell substrates that are readilyacceptable for use in human vaccine production, such as the humandiploid cell line MRC-5, in a medium virtually free of exogenous serumproteins. Surprisingly, under these conditions the cells continue toproduce PIV-3 for more than three weeks, entirely in the absence ofexogenously added growth factors. This process enables multiple virusharvests of similar antigenic composition to be obtained from the samegroup of cells. The absence of exogenous serum proteins greatlyfacilitates the process of purification.

Viral supernatants are either filtered or spun at low speed to removecellular debris and concentrated by ultrafiltration, when necessary. Thevirus then is pelleted by ultracentrifugation. The virus also can beisolated by passage of the ultrafiltration retentate over an affinitymatrix, such as Cellufine sulfate. The viral envelope glycoproteins thenare solubilized with an appropriate detergent (eg. Triton X-100 oroctylglucoside). Insoluble viral nucleocapsids are removed from thesolubilized material by centrifugation. We have shown that this step,while useful, need not be performed. The viral surface glycoproteins arepurified from the glycoprotein enriched fraction by affinitychromatography. Possible affinity matrixes include lentil-lectin andconcanavalin A covalently coupled to cross-linked Sepharose orcellulosic microporous membranes. Contaminating cellular and residualviral matrix proteins are eliminated in the flow-through and high saltwashes. Viral surface glycoproteins then are eluted from the column inthe presence of an appropriate competing sugar, such as methylα-D-mannopyranoside, in the presence or absence of salt. Highly purifiedglycoprotein preparations (as judged by Coomasie blue or silver stainedSDS polyacrylamide gels) are obtained using this process.

In accordance with the present process, HN and F from PIV-3 and F and Gproteins from RSV were affinity-purified. The PIV-3 HN and F proteinswere found to be highly immunogenic when tested in three separate animalmodels, namely guinea pigs, hamsters and cotton rats. Immunization ofanimals with varying doses of HN and F elicited a stronganti-glycoprotein antibody response. When administered with theappropriate adjuvants such as Freund's or aluminum phosphate, theminimal immunoprotective dose can be significantly reduced. Thus thefinal vaccine preparation when formulated with aluminum phosphate as anadjuvant can be used as a readily injectable preparation for human use.

The effectiveness of the invention is not only limited to thepreparation of the glycoproteins obtained from PIV-3 and RSV, but isapplicable to coat proteins from all paramyxoviridae. Our invention alsocovers the use of similar methods of isolation and the use of adjuvantsother than those mentioned.

EXAMPLES

Methods of determining hemagglutination (HA), tissue culture infectiousdose₅₀ (TCID₅₀), hemagglutination inhibition (HAI), neutralization andanti-fusion titres not explicitly described in this disclosure are amplyreported in the scientific literature and are well within the scope ofthose skilled in the art.

EXAMPLE I

This Example illustrates the production of PIV-3 by a mammalian cellline.

A stock of human PIV-3 virus was used to infect MRC-5 cells grown onmicrocarrier beads in a Bellco flask. A 35-litre culture of confluentMRC-5 cells grown in medium containing 10% fetal bovine serum wasdrained and the MRC-5 cells washed 3 times with 15 litres each of mediumCMRL 1969 containing 0.14% NaHCO₃. The cells were then infected withPIV-3 virus in a final volume of 10 litres of CMRL 1969 containing 0.14%NaHCO₃ and the virus allowed to adsorb to the cells for 2 hours at 37°C. with stirring. Following adsorption, an additional 25 litres ofmedium was added to the flask. These conditions reduced the serumproteins by an estimated 5,000 fold resulting in a final concentrationof less than 0.002%. The cells were incubated at 37° C. for 5 days andthe virus supernatant was collected. Approximately 35 litres of mediumCMRL 1969 containing 0.2% NaHCO₃ was added to the cells and the culturewas incubated for an additional 3 days and a second virus harvestobtained. An additional 35 litres of medium was then added and the cellsincubated a further 4 days prior to final harvest. Aliquots of all threevirus supernatants were assayed for infectivity and HA activity.Infectivity was determined in a standard TCID₅₀ assay using VERO cells,while HA activity was determined using guinea pig red blood cells at 37°C. The results, summarized in Table 1 below, clearly demonstrate thatMRC-5 cells produce substantial amounts of virus when cultured in therelative absence of exogenous serum proteins and that the same group ofMRC-5 cells are capable of providing three virus harvests eachcontaining substantial levels of virus. The process was alsosuccessfully scaled up to 150L bioreactors.

EXAMPLE II

This Example illustrates the preparation of purified PIV-3.

PIV-3 supernatant #2, obtained as detailed above, was processed usingtechniques readily amenable to large-scale vaccine production. The virussupernatant was first clarified by filtration. Tangential flowfiltration with a Sartorius Sartocon Mini unit incorporating 0.3 m² of0.45 um cellulose acetate membranes was used. Following clarification,virus was concentrated by tangential flow ultrafiltration using aMillipore Pellicon system incorporating 4 ft² of 100,000 nominalmolecular weight cutoff PTHK membranes. The Pellicon retentate then wasfiltered through a 0.22 um Millipore Millipak 20 unit, and viruspelleted by ultracentrifugation at 100,000×g for 1 hour at 4° C. Thepurified virus was resuspended in buffer. The HA and infectivity resultsare presented in Table 2 below. Essentially complete recovery of HAactivity and substantial recovery of virus infectivity was observedfollowing processing. These results demonstrate the suitability of thisprocess for PIV-3 purification.

EXAMPLE III

This Example illustrates the purification of PIV-3 HN and F proteins bylentil-lectin or concanavalin A Sepharose-4B affinity chromatography.

Pelleted virus, at a protein concentration of 1.5 mg/mL was treated atroom temperature for 1.5 hours with 2% v/v Triton X-100. Alternately,other detergents, such as octylglucoside or a combination of detergents(2% Triton X-100+2% w/v octylglucoside), can be used for proteinextraction. When necessary, these preparations are subsequently dialyzedagainst 0.02% Triton X-100. The insoluble nucleocapsid cores then wereremoved by centrifugation at 15000×g for 1 hour at 4° C. HN and Fproteins were purified from the glycoprotein-enriched supernatant byaffinity chromatography on either a lentil-lectin or concanavalin ASepharose column. The column first was washed with 10 bed volumes of PBScontaining 0.02% v/v Triton X-100. Following sample loading, the columnwas washed with 10 volumes PBS containing 0.02% Triton X-100. This wasfollowed by a high-salt wash consisting of 10 bed volumes of 0.35 M NaClcontaining 0.02% v/v Triton X-100 in PBS. The HN and F proteins wereeluted with 5 bed volumes of PBS containing 0.02% v/v Triton X-100 and0.3 M methyl-α-D-mannopyranoside. The eluted surface glycoproteins wereanalyzed by SDS-PAGE and found to be ≧95% pure by Coomasie blue andsilver staining of the polyacrylamide gels and scanning densitometry.

EXAMPLE IV

This Example illustrates the purification of RSV F and G proteins bylentil-lectin or concanavalin A Sepharose-4B.

Pelleted RSV (Long strain), at a protein concentration of approximately800 ug/mL, was treated at room temperature for 1.5 hours with 2% v/vTriton X-100. Insoluble nucleocapsid cores were removed bycentrifugation at 15000×g for 1 hour at 4° C. F and G proteins werepurified from the glycoprotein-enriched supernatant by affinitychromatography on either lentil-lectin or concanavalin A Sepharosecolumn. The procedure used for protein purification was essentiallyidentical to that used for purifying the PIV-3 HN and F proteins, asdetailed in Example III.

The eluted surface glycoproteins were analyzed by SDS-PAGE and found tobe >80% pure by silver staining of the polyacrylamide gels.

EXAMPLE V

This Example illustrates the immunogenicity of the lentil-lectinpurified PIV-3 glycoproteins in hamsters.

Four week old female hamsters (specific pathogen-free) were injectedintramuscularly with either 1.0, 0.5 or 0.1 ug of lentil-lectin purifiedHN and F proteins, prepared as described in Example III, administeredeither alone or in combination with aluminium phosphate or Freund'sadjuvants. Immediately after the 4 week bleed, animals were boosted withan equivalent dose of the antigen formulation. Sera samples also weretaken 6 and 8 weeks after the primary injection. HAI, neutralizing andanti-fusion titres were determined (summarized in Table 3 below). Datafrom the first bleed, (taken 4 weeks after the primary dose)demonstrated that all HN and F formulations tested can elicit a primaryimmune response. Inclusion of either aluminum phosphate or Freund'sadjuvants increased the HAI and neutralizing antibody responsesapproximately 10 and 30 fold, respectively. Animals responded in adose-dependent manner to immunization with 1.0, 0.5 or 0.1 ug ofadjuvanted antigen. Boosting the animals at 4 weeks with an equivalentdose of the antigen formulation resulted in a substantial increase inthe HAI, neutralizing and anti-fusion titres. At the 1 ug dose ofantigen, the immunopotentiating effects of both aluminum phosphate andFreund's adjuvants were evident. Thus, the immunogenicity of thelentil-lectin purified HN and F proteins has been clearly demonstrated.Antibody responses in guinea pigs and cotton rats were similar.

EXAMPLE VI

This Example illustrates the immunogenicity of the concanavalin Apurified PIV-3 glycoproteins in hamsters.

Hamsters (specific pathogen-free) were injected with either 1.0, 0.1,0.05, or 0.01 ug of concanavalin purified HN and F proteins, prepared asdescribed in Example III, in the presence of aluminum phosphate. Animalswere immunized according to the schedule outlined in Example IV. Animalsimmunized with adjuvanted concanavalin A purified proteins (the resultsare summarized in Table 4 below) responded in a dose-dependent manner toprimary injection with 1.0, 0.1, 0.05 or 0.01 ug of antigen. The minimalimmunogenic dose of adjuvanted concanavalin A purified proteins was 0.01ug. These results confirm the immunogenicity of the concanavalin Apurified HN and F proteins.

EXAMPLE VII

This Example illustrates the ability of the various HN and Fformulations to elicit a protective response in immunized hamsters andcotton rats.

Hamsters immunized with either 1.0, 0.5, 0.1, or 0.01 ug of thelentil-lectin purified HN and F preparations, prepared as described inExample III, were challenged with live PIV-3 virus immediately after the8 week bleed in order to evaluate the ability of the various HN and Fformulations to confer protective immunity. Hamsters were sacrificed 3days after challenge and their lungs removed and homogenized. Virus lungtitres are summarized in Table 5 below. Control animals injected withdiluted elution buffer supported the replication of 5.0 log₁₀ TCID₅₀units of virus per gram of lung tissue. In the absence of adjuvant, 75%of animals immunized with 1 ug of HN and F proteins had a detectablelevel of virus in their lungs. When HN and F was administered witheither aluminum phosphate or Freund's adjuvant, two 0.1 ug doses ofantigen protected all hamsters against live virus challenge. Virus wasnot detected in the lung homogenates. Similar results were obtained incotton rats.

Concanavalin A-purified HN and F proteins were also able to elicit aprotective response in immunized hamsters. Two 0.01 ug doses ofConcanavalin A purified proteins, prepared as described in Example III,(combined with aluminum phosphate) protected 80% of the animals againstlive virus challenge. These results demonstrate the ability of both thelentil-lectin and concanavalin A-purified HN and F proteins to protectthe lower respiratory tract of animals against live virus challenge.

The ability of the affinity-purified HN and F proteins to protect theupper respiratory tract of immunized animals against live viruschallenge was also tested. Virus nasal wash titers are summarized inTable below. The upper respiratory tract of animals immunized with two 1ug doses of adjuvanted Concanavalin A purified proteins, prepared asdescribed in Example III, was protected against PIV-3 infection. Viruswas not detected in nasal washes from this group of animals. This wasthe only group of animals tested which showed significant protection inthe upper respiratory tract following live virus challenge. Thus,concanavalin A-purified HN and F proteins, administered at a dose of 1ug plus aluminum phosphate, can evoke an immunological response capableof protecting both the upper and lower respiratory tracts of hamstersagainst live virus challenge.

EXAMPLE VIII

This Example illustrates that the various HN and F formulations do notcause “enhanced” histopathology in the lungs of immunized cotton ratsfollowing PIV-3 challenge.

Cotton rats were immunized with 1.0 or 0.1 ug of the lentil-lectinpurified HN and F proteins, prepared as described in Example III,administered either alone or with aluminum phosphate. Animals werechallenged intranasally with 100 median infectious doses of PIV-3immediately after the 8 week bleed. Four days after virus challenge,cotton rats were sacrificed and lung sections were prepared forhistopathological analysis. Lung sections from immunized and controlanimals were stained with hematoxylin and eosin, observed in a blindedfashion and evaluated for histopathology. The observed histopathologicalchanges generally correlated with lung virus titers and variedconversely with HAI and neutralizing antibody levels. Animals immunizedwith placebo and then challenged with PIV-3 had the most notablehistopathological changes. Sections of lung from these animals exhibitedmoderate to marked peribronchial and peribronchiolar lymphocyticinfiltration interspersed with polymorphonuclear leukocytes andmacrophages. Many of the bronchi and bronchioles observed were partiallydesquamified and the lumen of these passageways often contained amixture of debris, leukocytes and serous exudate. Scattered microareasof interstitial pneumonitis and perivascular cuffing were occasionallyseen. In contrast, pathological changes were minimal in animalsimmunized with protective doses of antigen. Most importantly, there wasno evidence of enhanced pathology in lung sections of any group ofimmunized animals when examined four days after virus challenge. In noinstance was the histopathology greater in immunized, challenged animalsthan in control animals immunized with placebo then challenged withPIV-3. It can, therefore, be concluded that the affinity-purified HN andF subunit vaccine has no short term immunopathological effects i.e.non-immunopotentiatiny.

TABLE 1 PI-3 Virus Production HA ACTIVITY TCID₅₀ ACTIVITY VOLUME HAU\Titer\ SAMPLE (ml) 0.5 ml Total ml Total Supernatant #1 34,000 32 2.2 ×10⁷ 10^(6.0) 5.4 × 10¹⁰ | Supernatant #2 34,000 32 2.2 × 10⁷ 10^(7.0)3.4 × 10¹¹ | Supernatant #3 25,000 16 8.0 × 10⁶ 10^(6.2) 4.0 × 10¹⁰ |

TABLE 2 Purification of PI-3 Virus HA ACTIVITY TCID₅₀ ACTIVITY TOTAL % %SAMPLE UNITS RECOVERY TOTAL RECOVERY Supernatant 2.2 × 10⁷ 100 3.4 ×10¹¹ 100  Clarified 2.2 × 10⁷ 100 2.1 × 10¹¹ 62 Retentate 2.0 × 10⁷  913.8 × 10¹⁰ 11 Filtered 1.9 × 10⁷  86 7.4 × 10¹⁰ 22 retentate 33k pellet2.5 × 10⁷ 114 4.8 × 10¹⁰ 14

TABLE 3 Serum Antibody Response of Hamsters Immunised with VariousLentil Lectin affinity-purified HN and F Formulations^(a) HAI^(b)Neutralisation^(c) Anti-Fusion^(d) Primary Secondary Primary SecondaryPrimary Secondary Antigen 4 Week 6 Week 8 Week 4 Week 6 Week 8 Week 4Week 6 Week 8 Week Formulation Dose Bleed Bleed Bleed Bleed Bleed BleedBleed Bleed Bleed Control — 10  10  10 10  10  10 4  4 4 HN & F alone1.0 11  80  47 10  37  24 4  4 4 HN & F + 1.0 100   640 640 79 645 457 816 8 AlPO₄ 0.5 50  645 457 56 513 457 4  8 8 0.1 28  575 575 28 407 4074  8 4 HN & F + 1.0 380  1819 1819  380  2570  1513  8 32 8 Freund's 0.5134  2560 1621  1123  2560  2041  4 64 32  0.1 40 1071 758 28 912 912 464 32  ^(a)= Each value represents the reciprocal geometric mean titrefrom 6 animals ^(b)= Serum dilution which inhibits erythrocyteagglutination by 4 hemagglutinating units of PIV-3 ^(c)= Serum dilutionwhich blocks hemadsorption of 100 TCID₅₀ units of PIV-3 ^(d)= Serumdilution which blocks syncytia formation by 100 TCID₅₀ units of PIV-3

TABLE 4 Serum Antibody Response of Hamsters Immunised with VariousLentil Lectin affinity purified HN and F Formulations HAI^(a)Neutralisation^(b) Primary Secondary Primary Secondary Antigen 4 Week 6Week 8 Week 4 Week 6 Week 8 Week Formulation Dose Bleed Bleed BleedBleed Bleed Bleed HN & F alone 1.0 70 320 160 68 240 320 HN & F + 1.0320  960 960 293  1920  1280  AlPO₄ 0.1 50 480 240 55 480 480 0.05 30267 200 25 373 200 0.01 35 256 224 28 288 176 ^(a)= Serum dilution whichinhibits erythrocyte agglutination by 4 hemagglutination units of Para-3virus. ^(b)= Serum dilution which blocks hemadsorption of 100 TCID₅₀units of Para-3 virus

TABLE 5 Response of Immunized Hamsters to PIV-3 Challenge^(a) (LowerRespiratory Tract) Dose of % Animals HN & F Antigen With virus MeanTitre ± SEM Formulation (ug) (b) log₁₀TCID₅₀ Elution buffer 100  4.9Lentil Lectin Purified HN and F alone 1.0 75  ≦4.1 HN and F + 1.0 0 ≦1.9Aluminium 0.5 0 ≦1.9 phosphate 0.1 0 ≦1.9 HN and F + 1.0 0 ≦1.9 Freund's0.5 0 ≦1.9 0.1 0 ≦1.9 Concanavilin A Purified HN and F alone 1.0 0 ≦1.9HN and F + 1.0 0 ≦1.9 Aluminium .1 0 ≦1.9 phosphate .01 20  1.9 ^(a) =Animals were challenged with 10⁶ TCID₅₀ units of PIV-3 and sacrificed 3days later. b = Minimum level of detectability was 10^(1.9) TCID₅₀/g oflung tissue. Each value represents the mean value of 6 animals.

TABLE 6 Response of Immunized Hamsters to PIV-3 Challenge^(a) (UpperRespiratory Tract) Dose of % Animals HN & F Antigen With virus MeanTitre ± SEM Formulation (ug) (b) log₁₀TCID₅₀ Elution buffer 100 5.0Lentil Lectin Purified HN and F alone 1.0 100 4.0 0.1 100 3.6 0.01 1003.5 HN and F + 0.1 100 4.2 Aluminium 0.01 100 4.2 phosphate HN and F +0.1 100 2.4 Freund's 0.01 100 4.7 Concanavilin A Purified HN and F alone1.0 100 4.0 HN and F + 1.0  0 ≦1.5 Aluminium .1 100 2.7 phosphate .01100 3.9 ^(a) = Animals were challenged with 10⁶ TCID₅₀ units of PIV-3and sacrificed 3 days later. b = Minimum level of detectability was10^(1.9) TCID₅₀/ml of nasal wash. Each value represents the mean valueof 6 animals.

SUMMARY OF DISCLOSURE

In summary of this disclosure, the present invention provides a novelprocedure for preparing immunogenic envelope glycoproteins as well asthe glycoproteins themselves. Modifications are possible within thescope of this invention.

What We claim is:
 1. A method of isolating a mixture of glycoproteins ofa virus belonging to the paramyxoviridae family, which comprises:growing a virus belonging to the paramyxoviridae family and selectedfrom the group consisting of respiratory syncytial virus (RSV) andparainfluenza virus (PIV), in a culture medium which is substantiallyfree from exogenous growth factors and serum proteins on a cellsubstrate which is readily acceptable for use in human vaccineproduction, isolating the virus from said culture medium, solubilizingthe glycoproteins from said isolated virus, and isolating andco-purifying said solubilized glycoproteins by affinity chromatographywherein the solubilized glycoproteins initially are bound tolentil-lectin or concanavilin A and subsequently eluted using acompeting sugar.
 2. The method of claim 1 wherein said cell substrate isthe human diploid cell line MRC-5.
 3. The method of claim 1 wherein saidvirus belonging to the paramyxoviridae family of virus is humanparainfluenza type 3 (PIV-3).
 4. The method of claim 3 wherein saidcopurified glycoproteins of PIV-3 are the glycoprotein HN andglycoprotein F.
 5. A composition, comprising a co-purified mixture ofviral envelope glycoproteins selected from the group consisting of amixture of the HN and F glycoprotein of parainfluenza virus (PIV) and amixture of the G and F glycoproteins of respiratory syncytial virus(RSV), and a carrier therefor.
 6. The composition of claim 5 comprisinga co-purified mixture of the glycoprotein HN and the glycoprotein F ofPIV-3.
 7. The composition of claim 5 for human administration containingaluminum phosphate as an adjuvant.
 8. The composition of claim 7formulated for administration by injection.
 9. The method of claim 1wherein said affinity chromatography is effected by lectin affinitychromatography.
 10. The method of claim 1 wherein multiple harvests ofviruses are taken from the same cell substrate.